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Cyclooxygenase-2 (COX) 2 promotes intestinal wound healing but elicits also proinflammatory effects and has been implicated in colorectal carcinogenesis. Thus, a balanced expression of COX-2 is essential for intestinal homeostasis. This study was designed to evaluate the regulation of COX-2 by probiotic organisms and to characterize ligands and receptors involved. Colo320 and SW480 intestinal epithelial cells (IEC) were stimulated with gastrin or TNF-alpha and pre- or coincubated with commensales, bacterial supernatants, or distinct toll-like receptor (TLR) ligands. COX-2 promoter activity was determined by luciferase assays, protein expression by Western blotting, and secretion of prostaglandin E(2) (PGE(2)) by ELISA. Commensales differentially regulated COX-2 expression in IEC. E. coli Nissle 1917, the probiotic mixture VSL#3, and media conditioned by these organisms ameliorated induced COX-2 expression and PGE(2) secretion. Heat inactivation and DNase treatment significantly decreased these regulatory capacities. Lactobacillus acidophilus, however, significantly increased COX-2 expression and PGE(2) secretion. TLR agonists differentially ameliorated basal or induced COX-2 expression. Distinct probiotics specifically and significantly decrease induced COX-2 expression in IEC, most likely mediated by released factors and in part by bacterial DNA. A significant involvement of TLRs in these regulatory processes remains to be established.